Metalloprotease disintegrin ADAM12 is emerging as an important breast cancer-related gene and a potential target for breast cancer therapies. Two somatic mutations, D301H in the metalloprotease domain and G479E in the disintegrin domain, are present in ~10% of primary breast tumors. We found that the D/H and G/E mutations lead to the retention of the nascent, inactive ADAM12 in the endoplasmic reticulum (ER) and a loss of active ADAM12 at the cell surface. The D/H and G/E mutants have a dominant-negative effect on wild-type ADAM12. The mechanisms responsible for the altered trafficking of the D/H and G/E mutants are not known. The main objectives of this proposal are to understand why the breast cancer-associated ADAM12 mutants are retained in the ER and what the consequences are of ADAM12 mislocalization in cancer cells. Our main hypothesis is that the D/H and G/E mutants are misfolded and retained by the ER quality control system, leading to ER stress and/or changing the repertoire of proteins secreted/shed to medium and the pattern of membrane-anchored proteins. The Specific Aims of this proposal are: 1. Determine why the D/H and G/E ADAM12 mutants are retained in the endoplasmic reticulum, and test approaches to restore the wild-type ADAM12 functionality to these mutants. 2. Assess the effect of the D/H and G/E ADAM12 mutants on the functional performance of the ER. 3. Compare the secretome and cell surface proteome of breast cancer cells expressing the wild-type ADAM12 and the D/H and G/E ADAM12 mutants. These studies will uncover the mechanism by which the breast cancer associated mutations change the function of ADAM12 in cancer cells. PUBLIC HEALTH RELEVANCE: This project is focused on the understanding the cause of malfunction of ADAM12 mutants that are frequently found in human breast tumors. The results of our studies will have a strong impact on breast cancer therapies tailored to patients harboring mutations in the ADAM12 gene.